Fig 1: HN1 promotes the self-renewal of BCSCs and migration and invasion of breast cancer cells. a Mammosphere formation analysis for the self-renewal ability of BCSCs with HN1 overexpression (*P <0.05). b SP analysis for the self-renewal ability of BCSCs with HN1 overexpression. c Flow cytometry analysis of the relative size of the CD24-CD44+ population after HN1 overexpression. d Wound healing assay showing the migration ability of MCF-7 and T47D with HN1 overexpression. e Transwell invasion analysis showing the invasion ability of indicated cells with HN1 overexpression (*P < 0.05). f Correlation of HN1 mRNA levels and the gene signatures about breast cancer relapse in brain using GSEA (*P <0.05). Each bar represents mean ± SD of 3 independent experiments
Fig 2: HN1 is upregulated in breast cancer tissues and correlates with poor prognosis according to clinical specimens. a Western blot assay of HN1 expression in normal breast tissues and breast cancer tissues. b IHC staining analysis of HN1 expression in patients with good prognosis and patients with poor prognosis. c Kaplan-Meier plots with log rank test for overall survival and relapse-free survival of breast cancer patients with high HN1 expression and low HN1 expression, respectively. Each bar represents mean ± SD of 3 independent experiments
Fig 3: Inhibition of MYC suppresses the phenotypes caused by HN1 overexpression. (a) Mammosphere formation analysis of the self-renewal ability of BCSCs. MCF-7 and T47D cells overexpressing HN1 were exposed to MYC-siRNA and MYC inhibitor 10058-F4 (*P <0.05). b Transwell invasion analysis showed the invasion ability of indicated cells. MCF-7 and T47D cells with overexpression of HN1 were exposed to MYC-siRNA and MYC inhibitor 10058-F4 (*P <0.05). Each bar represents mean ± SD of 3 independent experiments
Fig 4: HN1 is upregulated in breast cancer tissues and correlated with poor prognosis according to the data from TCGA database. a Dot plots represent HN1 expression levels in the primary breast cancer tissues (n = 1102) and normal breast tissues (n = 113) (**P <0.001). b HN1 expression levels in primary breast cancer tissues (n = 113) and matched adjacent normal breast tissues (**P <0.001). c Kaplan-Meier plots with log rank test for the overall survival (left), relapse free survival (middle) and distant metastasis free survival (right) of breast cancer patients with high HN1 expression and low HN1 expression, respectively (**P <0.001). d Quantitative real-time PCR analyzed HN1 expression in 12 breast cancer tissues and 4 normal breast tissues (*P <0.05)
Fig 5: HN1 regulates MYC expression. a Enrichment plot, indicating a significant correlation between HN1 mRNA level and the MYC regulated gene signatures (*P <0.05). b Correlation of MYC and HN1 expression (P = 0.044). c Western blot analysis of the expression of HN1 and MYC in indicated cells with HN1 overexpression or knockdown, ß-actin was used as the loading control. d The heatmap for the expression of MYC targeted genes including CDK4, CCND1, p21, CAV1, and SFRP1 after overexpression or knockdown of HN1. Quantitative real-time PCR was used to determine their expression. e Western blot analyzed the expression of CDK4, Cyclin D1 and p21 after HN1 overexpression or knockdown. GAPDH was used as the loading control. f Luciferase assay of MYC activity after overexpression or knockdown of HN1(*P <0.05). Each bar represents mean ± SD of 3 independent experiments
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